Hydroxylamine and its N- and O-methylated analogues are potent mutagenes which react with DNA to produce a variety of lesions. Cytosine is thought to be the primary mutagenic target but of the two (or three) cytosine adducts formed it is not clear which is the one responsible for the mutagenic event. The ratio of these products can be dramatically altered by changing pH, temperature, reagent concentration and more notably by the presence of a hydroxymethyl group at the C5 position of cytosine (C). The purpose of this study is (1) to determine whether alterations in the reaction products of hydroxylamines as predicted from available chemical data affect the rates of mutation and inactivation of bacteriophage T4; (2) to determine the frequency with which a hydroxylamine-modified residue in DNA mispairs in vivo; (3) to quantify the relative amounts of the products of reaction of hydroxylamine with T4 DNA containing either 5-hydroxymethylcytosine (5HMC) or C.